Kinetics of GLUT4 trafficking in rat and human skeletal muscle

Abstract

Objective: In skeletal muscle, insulin stimulates glucose transport activity three- to fourfold, and a large part of this stimulation is associated with a net translocation of GLUT4 from an intracellular compartment to the cell surface. We examined the extent to which insulin or the AMP-activated protein kinase activator AICAR can lead to a stimulation of the exocytosis limb of the GLUT4 translocation pathway and thereby account for the net increase in glucose transport activity.

Research design and methods: Using a biotinylated photoaffinity label, we tagged endogenous GLUT4 and studied the kinetics of exocytosis of the tagged protein in rat and human skeletal muscle in response to insulin or AICAR. Isolated epitrochlearis muscles were obtained from male Wistar rats. Vastus lateralis skeletal muscle strips were prepared from open muscle biopsies obtained from six healthy men (age 39 +/- 11 years and BMI 25.8 +/- 0.8 kg/m2).

Results: In rat epitrochlearis muscle, insulin exposure leads to a sixfold stimulation of the GLUT4 exocytosis rate (with basal and insulin-stimulated rate constants of 0.010 and 0.067 min(-1), respectively). In human vastus lateralis muscle, insulin stimulates GLUT4 translocation by a similar sixfold increase in the exocytosis rate constant (with basal and insulin-stimulated rate constants of 0.011 and 0.075 min(-1), respectively). In contrast, AICAR treatment does not markedly increase exocytosis in either rat or human muscle.

Conclusions: Insulin stimulation of the GLUT4 exocytosis rate constant is sufficient to account for most of the observed increase in glucose transport activity in rat and human muscle.

FIG. 1.

Comparison of the stimulation of glucose transport and cell-surface GLUT4 levels by insulin and AICAR in rat epitrochlearis muscle. A: Transport rates were determined from measurements of 3-O-methyl-d-[³H]-glucose uptake into muscle specimens incubated for the indicated times in the presence of 120 nmol/l insulin or 1 mmol/l AICAR. Results are means ± SE of six experiments. B: Levels of cell surface GLUT4 were determined by labeling muscle strips with GP15 photolabel following 30 min of incubation with 120 nmol/l insulin (■) and 50 min of incubation with 1 mmol/l AICAR (□). Labeled samples were solubilized, and the biotin-tagged GLUT4 was precipitated on immobilized streptavidin, resolved on SDS-PAGE gels, and then detected and quantified by blotting with GLUT4 antibody. Results are means ± SE of six (insulin) and five (AICAR) experiments.

FIG. 2.

Phospho-protein signaling in rat epitrochlearis and human vastus lateralis muscle. Immunoblots of p–Akt Ser473 (A), p–AMPK Thr172 (B), and p–CoA carboxylase Ser79 (C) in lysates of rat epitrochlearis and p–Akt Ser 473 (D) in human vastus lateralis muscle. Muscle lysates were resolved on SDS-PAGE, transferred to nitrocellulose membranes, and incubated overnight with phospho-specific antibodies. Immunoreactive proteins were visualized by enhanced chemiluminescence. Conditions are as follows: basal, initial basal; insulin 1, 12 nmol/l insulin stimulation for 52 min; recovery 1, 102-min recovery after removal of initial insulin stimulation; insulin 2, restimulation with 120 nmol/l insulin for 50 min after recovery; AICAR, restimulation with 1 mmol/l AICAR for 50 min after recovery; and recovery 2, 162-min recovery after removal of initial insulin stimulation. The figure includes representative immunoblots from six independent experiments.

FIG. 3.

Internalization of GP15-tagged GLUT4 under steady-state stimulation with insulin or AICAR in rat epitrochlearis muscle. Muscle strips were incubated with 120 nmol/l insulin or 1 mmol/l AICAR and then labeled with GP15. The GP15-tagged GLUT4 was then allowed to internalize for the indicated times. To separately identify internalized GLUT4, avidin was added to the medium to quench the signal from cell surface–located GLUT4. Internal GLUT4 was then solubilized, and the biotin-tagged GLUT4 was precipitated on immobilized streptavidin, resolved on SDS-PAGE gels, and detected and quantified by immunoblot analysis with a GLUT4 antibody. A: Representative blots. init, initial labeling without avidin addition. B: By comparison of the initial GLUT4 signal with the signal at the indicated times, the level of cell-surface GLUT4 was calculated. C and D: Curve fitting to data from separate experiments was used to calculate rate constants as described in research design and methods. The curves in B were computed from the average rate constants in C. Results are the means ± SE of four experiments. *P < 0.05.

FIG. 4.

Exocytosis of GP15-tagged GLUT4 in rat epitrochlearis muscle. Muscle specimens were incubated with 12 nmol/l insulin and labeled with GP15. Insulin action was then terminated by washing with a pH 6.0 buffer, and the tagged GLUT4 was allowed to internalize for 40 min. Muscle specimens were maintained in the basal state or stimulated with either 120 nmol/l insulin or 1 mmol/l AICAR for the indicated times. Avidin was added at the cell surface throughout the restimulation time courses; this reduced the signal for GLUT4 that exocytosed to the cell surface, thereby reducing the signal from internal GLUT4. The remaining internal GLUT4 was then solubilized, and the biotin-tagged GLUT4 was precipitated on immobilized streptavidin, resolved on SDS-PAGE gels, and detected and quantified by immunoblot analysis with a GLUT4 antibody. A: Representative blots. C: Curve fitting for internal GLUT4 was carried out for the basal, insulin, and AICAR treatments and data from separate experiments were used to calculate rate constants as described in research design and methods. B: The curves were computed from the average rate constants in C. Results are means ± SE. Data are derived from seven, five, and three experiments for basal, insulin, and AICAR treatments, respectively. Basal experiments had at least three time points, but these were varied to allow comparison with early and late time points for the insulin or AICAR stimulations. *P < 0.05.

FIG. 5.

Exocytosis of GP15-tagged GLUT4 in human vastus lateralis muscle. Muscle strips were incubated with 12 nmol/l insulin and then labeled with GP15. Insulin action was then terminated by washing with a pH 6.0 buffer, and the tagged GLUT4 was allowed to internalize for 40 min. The strips were then maintained in the basal state or stimulated with either 120 nmol/l insulin or 1 mmol/l AICAR for the indicated times. Avidin was added at the cell surface throughout the restimulation time courses; this reduced the signal for GLUT4 that exocytosed to the cell surface, thereby reducing the signal from internal GLUT4. The remaining internal GLUT4 was then solubilized and the biotin-tagged GLUT4 was precipitated on immobilized streptavidin, resolved on SDS-PAGE gels, and detected and quantified by immunoblot analysis with a GLUT4 antibody. A: Representative blots. C: Curve fitting was carried out for the basal, insulin, and AICAR treatments, and data from separate experiments were used to calculate rate constants as described in research design and methods. B: The curves were computed from the average rate constants in C. Results are means ± SE. Where no error bar is shown, n = 2. Data are derived from four, four, and two experiments for basal, insulin, or AICAR treatment, respectively. In basal experiments, time points were varied to allow comparison with early and late time points for the insulin and AICAR stimulations. *P < 0.05.